The central goal of the experiments in this proposal is to understand how neurons find their targets during neural development. Our current understanding of how various guidance cues and their receptors influence the establishment and maintenance of neuronal trajectories provides a platform upon which a thorough understanding of the signaling cascades which govern neuronal guidance can be built. The semaphorin family of phylogenetically conserved proteins contains many well characterized repulsive guidance cues. However, some semaphorins can act as attractants, and certain individual semaphorins have the capacity to act as both a repellent and an attractant. Therefore, defining the semaphorin signaling cascade will contribute to our understanding of how repulsive guidance is signaled to neuronal processes and also how this signaling is modulated. In addition to aiding our understanding of how growth cones navigate through complex extracellular environments during neural development, elucidation of semaphorin signaling cascades has important clinical implications. Semaphorin signaling has been linked to inhibition of neuronal extension following injury, to the progression of certain cancers, and to immune system function. Therefore, work proposed here has potential implications that extend beyond understanding how neuronal connectivity is established and maintained. We propose here to investigate how key semaphorin signaling cascade components steer neuronal processes. We have previously characterized two phylogenetically conserved different protein families, the MICALs and nervy/MTG proteins, each of which includes cytosolic proteins that play critical, but distinct, roles in promoting and modulating semaphorin-mediated repulsion in vivo. This work provides new insights for our current understanding of semaphorin signaling which can be applied to both invertebrate and vertebrate guidance events. Therefore, using approaches in both Drosophila and in rodents, we will address how these and other semaphorin signaling cascade intermediates ultimately facilitate the establishment and maintenance of neuronal connectivity.